Malignant pleural mesothelioma (MPM) is a neoplasm for which no effective therapy currently exists. We completed a Phase I clinical trial involving intrapleural delivery of an E1/E3-deleted adenovirus (Ad) containing the HSVtk gene (H5.010RSVtk), followed by systemic treatment with ganciclovir (GCV) in 26 patients with MPM. Treatment at dose levels up to 5x1013 viral particles revealed evidence of dose-dependent, but superficial, intratumoral tk gene transfer, as well as significant immune responses to the Ad vector and the tk protein. Minimal toxicities were seen and no maximally tolerated dose (MTD) of H5.010RSVtk was established. In order to improve intratumoral gene transfer, we have initiated a new Phase I trial using a "third generation" E1/E4-deleted adenoviral vector (H5.001RSV.tk) that, in animal models, is equally effective, but less immunogenic and hepatotoxic. This new vector is also more cost-efficient to produce because of a lower incidence of homologous recombination. We have treated four patients with the E1/E4-deleted adenovirus to date, two each at 1.5x1013 and 5x1013 viral particles. Intratumoral gene transfer has been detected via immunohistochemistry in all 4 patients. Toxicities have been limited to transitory pyrexia after vector instillation and minimal elevation of transaminases. We plan to continue the dose-escalation protocol until a MTD is established. Determination of the immune response to vector and transgene is ongoing, as is evaluation of tumor response using volumetric Chest CT and 18-FDG PET imaging. In summary, Ad.HSVtk /GCV gene therapy with the E1/E4-deleted vector is safe in MPM patients to doses of 5x1013 viral particles, with evidence of tk gene transfer in a dose-dependent fashion. The clinical trial is supported by NIH PO1CA66726.